Tissue Cryopreservation Research Articles

Research Progress of Ovarian Tissue Cryopreservation and Transplantation

Age, disease and anti-tumor treatment may cause the decline or even loss of female fertility. Embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation are commonly used at present. Cryopreservation and transplantation of ovarian tissue has become a fertility preservation method with medical indications in clinic, which is helpful for patients to successfully complete fertility through this technology. This paper reviews the international research progress on ovarian cryopreservation and transplantation in recent years. Despite the rapid development of ovarian tissue cryopreservation and transplantation technology, it still faces challenges such as how to reduce the loss of follicular reserve and thus prolong the effectiveness of ovarian tissue transplantation. More basic and clinical research is needed to promote the progress and development of ovarian tissue cryopreservation and transplantation technology.

Melatonin increases superoxide dismutase 2 (SOD2) levels and improves rat ovarian graft function after transplantation

BackgroundOvarian cryopreservation is a promising technique despite being hindered by damage from freezing and thawing. Melatonin can mitigate this outcome.ObjectiveThis study aimed to evaluate the effect of melatonin on the follicular dynamics of ovarian tissue in a cryopreserved cell culture.MethodsThree-month-old adult female Wistar rats (n = 24) weighing approximately 250 g were oophorectomized and divided into two groups (n = 12): the control group (CG) and the melatonin group (MG). In the CG, slow cryopreservation was performed according to the standard protocol with Medium M2 and dimethyl sulfoxide (DMSO). In MG, melatonin diluted in ethyl alcohol vehicle at a concentration of 0.1 μm was added to the culture medium. In both groups, the ovaries were cryopreserved by slow freezing and kept in liquid nitrogen for 24 h. Subsequently, after thawing, the ovaries were reimplanted in the retroperitoneum, one on each side of the great vessels (inferior vena cava and aorta). After 30 days, the animals were euthanized during the diestrus phase; then, the grafts were removed and processed for histomorphometric and immunohistochemical analyses, whereas the blood was subjected to biochemical analysis. Student’s t test was used to assess the difference between the groups.ResultsThe FSH levels in MG (83.79 ± 32.37) were lower than those in CG (120.52 ± 36.59), p = 0.03. The FSH/AMH ratios were also lower in MG (3.53 ± 1.13) than in CG (6.52 ± 2.85), p = 0.001. The SOD2 immunoexpression was higher in MG than in CG regarding all parameters except for the degenerated follicles (follicular cells and internal thecal cells): CG (16.80 ± 4.80 [13.36–20.24]) and MG (14.91 ± 4.04 [12.01–17.79]) p = 0.351. Statistically, the difference in intact follicles (theca + interstitium) between CG (6.60 ± 2.59 [4.75–8.45]) and MG (9.31 ± 3.09 [7.09–11.51]) was significant (p = 0.049), with a small difference in the expression of regular antral follicles.ConclusionsMelatonin can improve the quality of cryopreserved tissues, as evidenced in this study, and the evaluation of cryopreserved ovarian grafts, as shown in the melatonin group with better hormonal parameters and greater immunohistochemical expression of the SOD2 antioxidant. Thus, damage is reduced during cryopreservation and transplantation is improved.

Autologous Cryopreserved Adipose Tissue Using an Innovative Technique: An In Vitro Biological Characterization

Abstract Background Utilization of autologous adipose tissue transplantation in plastic and orthopedic surgery such as breast reconstruction and intra-articular injection has become an attractive surgical treatment with satisfactory clinical outcomes. Nevertheless, repeated liposuctions necessary to harvest fatty tissue, normally performed with sedation or general anesthesia, may represent a noteworthy concern. Objectives The aim of this study was to demonstrate through an in vitro characterization the validity of the surgical option of cryopreserved autologous adipose tissue harvested in a single shot for repeated graft transfer in breast reconstruction without impairment of cell viability and sterility. Methods Adipose tissue was collected by standard liposuction from patients who needed numerous fat grafting procedures for breast reconstruction. According to an innovative and patented cryopreservation method, autologous adipose tissue was subsequently fractioned in a sterile bag system and frozen at the RER Tissue Bank of the Emilia Romagna Region. Each graft was evaluated for sterility and cell viability immediately after harvesting, and 1, 3, 6, 12, and preliminarily 18 months after cryopreservation and thawing. Results In vitro results showed that after processing, middle-term and long-term cryopreservation, and subsequent thawing, autologous cryopreserved adipose tissue retained absence of bacterial contamination, high cellular viability, and unmodified histomorphological properties, thereby ensuring maintenance of the stromal vascular niche and the filling properties in different multistep surgical procedures. Conclusions In vitro study and sterility assessment showed that autologous cryopreserved adipose tissue grafting is a safe procedure, making it possible to avoid multiple liposuction surgery. No impairment of sterility, cell viability, or morphology was observed over time.

Data-driven discovery of potent small molecule ice recrystallisation inhibitors

Controlling the formation and growth of ice is essential to successfully cryopreserve cells, tissues and biologics. Current efforts to identify materials capable of modulating ice growth are guided by iterative changes and human intuition, with a major focus on proteins and polymers. With limited data, the discovery pipeline is constrained by a poor understanding of the mechanisms and the underlying structure-activity relationships. In this work, this barrier is overcome by constructing machine learning models capable of predicting the ice recrystallisation inhibition activity of small molecules. We generate a new dataset via experimental measurements of ice growth, then harness predictive models combining state-of-the-art descriptors with domain-specific features derived from molecular simulations. The models accurately identify potent small molecule ice recrystallisation inhibitors within a commercial compound library. Identified hits can also mitigate cellular damage during transient warming events in cryopreserved red blood cells, demonstrating how data-driven approaches can be used to discover innovative cryoprotectants and enable next-generation cryopreservation solutions for the cold chain.

Pathways to motherhood: A single-center retrospective study on fertility preservation and reproductive outcomes in patients with breast cancer

BackgroundBreast cancer treatments often have negative effects on fertility, which pose challenges among patients who want to be parents in the future. This study aimed to examine the efficacy of oocyte cryopreservation, embryo cryopreservation, and ovarian tissue cryopreservation in patients with breast cancer. MethodsThis retrospective review evaluated 42 patients with breast cancer who underwent fertility preservation at our center from January 2012 to December 2022. This review encompassed the demographic characteristics of the patients, cancer stages, treatment details, and types of fertility preservation procedures and their outcomes. ResultsThe average age at disease diagnosis was 33.4 years. Approximately 90.4% of patients presented with early-stage cancer (≤2). Of 42 patients, 26 underwent oocyte cryopreservation; 17, embryo cryopreservation; and 2, ovarian tissue cryopreservation. Further, three patients received mixed treatment. The overall live birth rate was 63.2%. There are more live births in embryo cryopreservation group. The successful pregnancy group was significantly younger and had a remarkably higher quantity of preserved oocytes/embryos than the nonsuccessful pregnancy group. The oocyte and embryo utilization rates in cryopreservation were 7.69% and 52.94%, respectively. These findings underscored the importance of prompt, informed discussions about fertility preservation options. ConclusionFertility preservation in patients with breast cancer have promising reproductive outcomes, with embryo cryopreservation being particularly effective. Prompt counseling and individualized fertility preservation strategies are important for improving the likelihood of posttreatment pregnancy. Nevertheless, future research on the long-term psychological and emotional effects of different fertility preservation methods must be performed.

Is Routine Pathology Evaluation of Tissue Removed for Fertility Preservation Necessary?

BackgroundFor all fertility preservation (FP) cases at our institution, a biopsy is performed for routine pathology from all gonadal tissue removed. This is not standard at all centers. We reviewed our experience with biopsy for pathological evaluation of ovarian and testicular specimens in FP cases to determine clinical utility. MethodsThe medical records of individuals who underwent ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) between 2011 and 2023 were retrospectively reviewed under an IRB-approved study at a free-standing tertiary care children's hospital. Patient demographics, diagnosis, operative characteristics, and pathology results were collected. ResultsOne-hundred and eighty-three patients underwent OTC, and 134 patients underwent TTC. All patients had their gonadal tissue biopsied for routine pathology. Malignancy was identified in the biopsies of 4 OTC patients (2.2%) and 2 TTC patients (1.5%). Two OTC patients (1.1%) and 2 TTC patients (1.5%) did not have germ cells identified in their biopsy. All OTC and TTC patients and families elected to continue storing tissue for FP after discussion of pathology findings. ConclusionsPathology results provide another data point to help inform patients and their families when making decisions on ovarian or testicular tissue storage and on how tissue may be utilized in the future to restore fertility and/or hormones. There is a low rate of identifying malignancy in gonadal tissue biopsies taken from FP specimens even in patients with known malignancy. However, when malignancy was identified, it could be unexpected and alter the diagnosis and treatment plan significantly for patients. Level of EvidenceIV.

P-220 Two strategies for reducing cryoprotectant toxicity: recent trends in oocyte, embryo and tissue cryopreservation

Abstract Study question In this study, we demonstrate two methods to reduce the toxicity of cryoprotectants. Both have been tested in cells and tissues Summary answer Employing both the theoretical model of the incubation process and an ultra-rapid rewarming based on high-intensity focused ultrasound, it is possible to reduce this toxicity What is known already Cryoprotectants used in assisted reproduction for the cryopreservation of gametes, embryos, or ovarian or testicular tissue are based on DMSO, ethylene glycol, glycerol, etc. ... Minimizing exposure to these substances is always desirable. In this work, we show how, by modeling the hydraulic permeability of the embryo membrane and by the practical application of high-intensity focused ultrasound, it is possible to drastically reduce the biological material’s exposure to these agents. The former achieves a 90% reduction in incubation times in the cryoprotectant. The latter significantly increases the speed of rewarming, thereby reducing the amount of cryoprotectant needed for tissue recovery. Study design, size, duration The system was modeled using computer simulations based on MATLAB algorithms. It has been verified that in 1 minute, 90% of the cryoprotectant penetrates the embryo. The results have been experimentally validated in human embryos and oocytes. Regarding rewarming, High-Intensity Focused Ultrasound (HIFU) has achieved rewarming rates two orders of magnitude higher than standard rewarming. This has enabled the recovery of tissue samples that otherwise would not have been viable. Participants/materials, setting, methods For this study, donated oocytes and embryos were used. The MATLAB programming language was utilized for computer simulations. For ultrasonic rewarming, a piezoelectric excited at a frequency of 1.2 MHz and 60VDC was used. Standard vitrification was compared with our toxicity reduced protocol in the case of oocytes and embryos, Standard slow freezing was used in the case of rewarming with high intensity focused ultrasound. Main results and the role of chance The application of mathematical modeling, together with the experimental resultas on oocytes and embryos, demonstrate that is it possible to reduce in 90% the toxicity of the cryoprotectant in a typical vitrification protocol. Also, the application of High Intensity Focused Ultrasound reaches warming rates that are two orders of magnitud higher than the conventional rewarming in a water bath. This finding represent a significative improvement of the present cryopreservation protocols. The success of both strategies is demonstrated, both in reducing incubation time and the superiority of rewarming using ultrasound. The study is generally applicable to oocytes, sperm, embryos, ovarian and testicular tissue. Limitations, reasons for caution No limitations. Wider implications of the findings We believe that these findings will soon be adopted by centers where the cryopreservation of biological material is necessary. Trial registration number NOT APPLICABLE

O-245 Is AMH testing relevant for fertility preservation in young oncology patients?

Abstract As the prevalence of cancer in young people rises, and with improved survival rates such that over 80% of young cancer patients can expect to recover, future fertility and having their own family has become an important consideration which needs to be addressed. Best-practice oncofertility management includes initial discussion, assessment of fertility risk and medical risk, and consideration of preservation strategies prior to commencement of potentially gonadotoxic therapy, where possible, as well as post cancer-treatment fertility care. The window of opportunity for oocyte cryopreservation and/or ovarian tissue cryopreservation may be very limited. Therefore it is of paramount importance that a patient’s medical condition, gonadotoxicity risk, and current reproductive health and potential are considered when planning fertility preservation. Antimullerian hormone (AMH) is one of a group of tests performed which can assist with planning treatment and with counselling regarding the likely yield of a cycle. Knowledge regarding pre-treatment AMH, along with a patient’s age and the type of cancer treatment, may also assist with counselling regarding the likelihood of recovery of fertility after completion of this treatment. However, there are many factors which affect AMH levels, and it is well-known that the results of stimulation can be very variable, as can be the long-term impact of the potentially gonadotoxic treatment. Hence an AMH test, while useful, needs to be contextualized as being only one of many factors to be considered when planning optimal (in terms of yield and safety) fertility preservation treatment and advising re future fertility.

P-325 Comparing slow-freezing and vitrification endometrial biopsy cryopreservation protocols for tissue cryodamage and functional organoid formation

Abstract Study question Are slow-freezing and vitrification suitable methods for cryopreserving endometrial biopsies intended for downstream research applications and organoid formation? Summary answer Both slow-frozen and vitrified endometrial biopsies resulted in successful organoid formation thus can be considered similar to fresh endometrial biopsies in preserving the cellular viability. What is known already Passive slow freezing (PSF) protocols have demonstrated efficacy for cryopreserving endometrial biopsies with subsequently viable isolated epithelial and stromal cells. Additionally, single-cell transcriptomic analyses and successful generation of endometrial organoids suggest a little impact of cryopreservation. By avoiding ice crystal formation, vitrification (VT) could potentially prevent mechanical injury and improve tissue integrity compared to slow freezing. Study design, size, duration Experimental study using endometrial biopsies collected using a Pipelle from eight healthy volunteers. Samples were retrieved at three cycle phases: proliferative (n = 2), mid-secretory (7 days post-LH surge, LH + 7, n = 2) and late-secretory phase (LH + 11, n = 4). Each biopsy was divided into fresh, slow-freezing and vitrification sub-samples for further comparison of outcomes according to microscopic histological analysis, transmission electron microscopy (TEM), gene expression profiling evaluation and generation of endometrial epithelial organoids (from 2 slow-frozen and 2 vitrified samples). Participants/materials, setting, methods PSF was performed using Mr Frosty with samples embedded in 1x DMEM, 30% FBS and 7.5% DMSO at -80ºC. Vitrification was performed using 40% ethylene glycol (EG); 30% Ficoll; 0.5 M Sucrose and 10 mg/ml HSA, and preserved in liquid nitrogen. Tissue morphology was evaluated by traditional histology, TEM and gene expression profiling. The viability of endometrial cells and the ability to form epithelial organoids were evaluated before and after two different freezing protocols. Main results and the role of chance Our analysis showed that both PSF and vitrification techniques are appropriate for cryopreservation and storage of endometrial biopsies aimed at organoid generation, as no significant differences were found between fresh and cryopreserved tissue samples. However, ultrastructural changes were observed by TEM in PSF samples indicating an impact on mitochondria of epithelial and stromal cells with increased swelling. Changes were also observed in epithelial cells in vitrified samples, with decreased nuclear granularity, clarity, and open karyoplasm. On TEM analyses changes in the chromatin status were more evident in vitrified samples compared to fresh and PSF samples. PSF treated samples showed similarly high quality of epithelial and stromal nuclear staining when compared to the fresh samples in haematoxylin and eosin-stained slides, while VT of samples resulted in a significant decrease of both epithelial and stromal nuclear clarity. However, despite these changes observed in the tissue morphology, all three groups displayed similar transcriptional profiles and endometrial organoids were generated from both slow-frozen and vitrified samples, indicating very little or no impact of the freezing method used for post-thaw tissue functionality. Limitations, reasons for caution Although our study demonstrated relatively mild damages in both freezing protocols, we cannot exclude the absence of additional changes not evaluated in this study, such as those concerning the epigenetic constitution of the cells. Therefore, more extended research with larger sample size is needed for further validation of the results. Wider implications of the findings Our findings indicate that both slow-freezing and vitrification methods applied to endometrial biopsies are effective in maintaining tissue morphology and functionality. Further optimization of cryopreservation methods of endometrium could contribute to future experimental and clinical use of cryopreserved endometrial tissue. Trial registration number not applicable

Melatonin activates Nrf2/HO-1 signalling pathway to antagonizes oxidative stress-induced injury via melatonin receptor 1 (MT1) in cryopreserved mice ovarian tissue.

Our previous research has shown that melatonin (MLT) can reduce cryopreserved ovarian damage in mice. Yet, the molecular mechanism of MLT protection is still unclear. Some studies have shown that melatonin receptor 1 (MT1) is very important for animal reproductive system. To evaluate whether MLT exerts its protective effect on cryopreserved mice ovarian tissue via MT1, we added antagonist of MT1/MT2 (Luzindor) or antagonist of MT2 (4P-PDOT) to the freezing solution, followed by cryopreservation and thawing of ovarian tissue. The levels of total superoxide dismutase (T-SOD), catalase (CAT), nitric oxide (NO) and malondialdehyde (MDA) were detected. Besides, by using RT-PCR and Western blotting, the expression of Bcl-2, Bax and Nrf2/HO-1 signalling pathway-related proteins was detected. These findings demonstrated that compared with the melatonin group, the addition of Luzindor increased apoptosis, NO and MDA activities, decreased CAT and T-SOD activities and inhibited Nrf2/HO-1 signalling pathway. In conclusion, melatonin can play a protective role in cryopreserved ovarian tissue of mice through MT1 receptor.

Cryopreservation of human adult ventricular tissue for the preparation of viable myocardial slices

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): REGeRNA Myocardial slices are ultra-thin slices of living adult myocardium which can be cultured. As a multicellular 3-D culture model, myocardial slices offer a novel opportunity to investigate how adult cardiomyocytes (CMs) interact with other cardiac cell types and the extracellular matrix, while providing long-term preservation of CMs cytoarchitecture and electrical phenotype. Additionally, myocardial slices are a new tool in drug discovery by allowing to test chronic treatments, as opposed to isolated CMs which are limited to acute timepoints. Despite their benefits over other cardiac preparations, myocardial slices also have several limitations, of which one is the quality of the slices. The main determinant of myocardial slice quality is the procedure by which the cardiac tissue block is handled. Current methods depend on the preparation of slices directly after tissue excision, therefore limiting the moment when the experiment is performed. We developed a protocol for the cryopreservation of human adult ventricular tissue allowing preparation of viable myocardial slices afterwards. Fresh and cryopreserved cardiac tissue from donor hearts was processed and evaluated for viability, tissue integrity, and inflammatory markers using molecular biology and histological assays. The tissue samples retained viability, biomass, and tissue and vascular integrity following cryopreservation compared to fresh tissue. Additionally, we compared myocardial slices obtained from fresh or cryopreserved tissue and cultured for 24 hours. Histologically, normal cytomorphological features and gap junctions between CMs were preserved in all slices. Comparable retention of tissue viability and structural integrity post cryopreservation (4-6 weeks) and culture (up to 7 days) was observed in cardiac samples from 3 additional donors. In summary, cold storage extends the time-interval between sample collection and slice preparation, allowing for more long-term, complex endpoints. Moreover, efficient banking of cryopreserved cardiac samples from various donors and disease states is critical for regular use of myocardial tissue slices in drug discovery and development. Additional research on the effects of cryopreservation on different cardiac cell types will allow to improve donor organ management to extend the donor pool and increase utilization rate.

Good Manufacturing Practice-Compliant Cryopreserved and Thawed Native Adipose Tissue Ready for Fat Grafting.

Background: As adipose tissue-derived mesenchymal stem cells are becoming the tool of choice for many clinical applications; standardized cryopreservation protocols are necessary to deliver high-quality samples. For this purpose, the cryopreservation and thawing of native adipose tissue under GMP conditions could represent an extremely useful and powerful tool for the direct reinfusion of the tissue, and consequently, of its stromal vascular fraction. Methods: In this study, 19 samples of adipose tissue were cryopreserved and characterized before and after storage in liquid nitrogen vapors. Of these 19 samples, 14 were processed in research and 5 in a GMP-compliant environment. Storage with and without cryopreservation medium was also evaluated. After one week to three months of storage, samples were thawed, washed, enzymatically digested, and characterized with flow cytometry. Results: The results show that there is a loss of nearly 50% of total nucleated cells during the cryopreservation/thawing process. Non-GMP and GMP samples are comparable for all parameters analyzed. This study also allowed us to exclude the cryopreservation of adipose tissue without any cryopreservation medium. Conclusions: The data shown in this work are consistent with the idea that native adipose tissue, if properly processed and controlled, could be a useful source of cells for regenerative medicine, keeping in mind that there is a clear difference in the quality between fresh and thawed samples.

Reproductive Strategies for Declining Fertility: Fertility Preservation

There is a global trend of declining fertility among people of childbearing age and mankind is confronted with great challenges of fertility problems. As a result, fertility preservation technology has emerged. Fertility preservation involves interventions and procedures aimed at preserving the patients' chances of having children when their fertility may have been impaired by their medical conditions or the treatments thereof, for example, chemotherapy and/or radiotherapy for cancer. The changes in patients' fertility can be temporary or permanent damage. Fertility preservation can help people diagnosed with cancer or other non-malignant diseases. More and more fertility preservation methods are being used to preserve the fertility of cancer patients and protect their reproductive organs from gonadotoxicity. Fertility preservation may be appropriate for young patients with early-stage cancers and good prognosis before they undergo treatments (chemotherapy and/or radiotherapy) that can negatively affect their fertility. It is also appropriate for patients with chronic conditions or those who have encountered environmental exposures that affect their gonadal function. Fertility preservation methods include oocyte cryopreservation, embryo cryopreservation, and ovarian tissue cryopreservation (OTC) for women and sperm freezing and testicular tissue freezing for men. The survival rates of children and adolescents diagnosed with malignant tumors have been steadily increasing as a result of advances in cancer treatments. Cryopreservation of oocytes and sperm is recognized as a well-established and successful strategy for fertility preservation in pubertal patients. OTC is the sole option for prepubertal girls. On the other hand, cryopreservation of immature testicular tissue remains the only alternative for prepubertal boys, but the technology is still in the experimental stage. A review showed that the utilization rate of cryopreserved semen ranged from 2.6% to 21.5%. In the case of cryopreserved female reproductive materials, the utilization rate ranged from 3.1% to 8.7% for oocytes, approximately from 9% to 22.4% for embryos, and from 6.9% to 30.3% for ovarian tissue. When patients have needs for fertility treatment, cryopreserved vitrified oocytes are resuscitated and in vitro fertilization-embryo transfer (IVF-ET) was performed to help patients accomplish their reproductive objectives, with the live birth rate (LBR) being 32%. On the other hand, when cryopreserved embryos are resuscitated and transferred, the LBR was 41%. OTC has the advantage of restoring natural fertility and presents a LBR of 33%, compared with the LBR of 19% among 266 IVF patients. In addition, OTC has the benefit of restoring the endocrine function. It has been observed that the shortest recovery time of the first menstruation after transplantation was 3.9 months, and the recovery rate of ovarian function reached 100%. To date, a growing number of cancer survivors and patients with other diseases are benefiting from fertility preservation measures. In the face of declining human fertility, fertility preservation provides a new approach to human reproduction. Fertility preservation should be applied in line with the ethical principles so as to fully protect the rights and interests of patients and their offsprings.

Review of the Low-Temperature Acoustic Properties of Water, Aqueous Solutions, Lipids, and Soft Biological Tissues.

Existing data on the acoustic properties of low-temperature biological materials is limited and widely dispersed across fields. This makes it difficult to employ this information in the development of ultrasound applications in the medical field, such as cryosurgery and rewarming of cryopreserved tissues. In this review, the low-temperature acoustic properties of biological materials, and the measurement methods used to acquire them were collected from a range of scientific fields. The measurements were reviewed from the acoustic setup to thermal methodologies for samples preparation, temperature monitoring, and system insulation. The collected data contain the longitudinal and shear velocity, and attenuation coefficient of biological soft tissues and biologically relevant substances-water, aqueous solutions, and lipids-in the temperature range down to -50 °C and in the frequency range from 108 kHz to 25 MHz. The multiple reflection method (MRM) was found to be the preferred method for low-temperature samples, with a buffer rod inserted between the transducer and sample to avoid direct contact. Longitudinal velocity changes are observed through the phase transition zone, which is sharp in pure water, and occurs more slowly and at lower temperatures with added solutes. Lipids show longer transition zones with smaller sound velocity changes; with the longitudinal velocity changes observed during phase transition in tissues lying between these two extremes. More general conclusions on the shear velocity and attenuation coefficient at low-temperatures are restricted by the limited data. This review enhance knowledge guiding for further development of ultrasound applications in low-temperature biomedical fields, and may help to increase the precision and standardization of low-temperature acoustic property measurements.

Optimal human ovarian follicle isolation: A review focused on enzymatic digestion.

The damage or depletion of ovarian reserves due to aging or cancer treatment can increase the need for fertility preservation techniques. One of the most common ways of supporting fertility in prepubertal girls and women who require immediate cancer treatment is through ovarian tissue cryopreservation and re-transplantation following cancer treatment. However, a more appropriate method should be employed in diseases such as leukemia, where malignant cells may be present in cryopreserved tissue, instead of ovarian tissue transplantation. Human ovarian follicle isolation for in vitro culture or the use of artificial ovaries for their growth can decrease the risk of reintroducing cancer cells into these individuals. Here we review the methods for the isolation of human ovarian follicles.

Abstract 4957: DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue

Abstract Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing of tumor tissue in clinical cancer care comes primarily from formalin-fixed, paraffin-embedded tissue (FFPE). Specific to genomics assays, numerous studies have shown significant discordance in genetic information obtained from FFPE samples and frozen samples. To explain the discordance between FFPE samples and cryopreserved samples, a head-to-head comparison between FFPE and cryopreserved tissues was performed to analyze the DNA yield, DNA purity and DNA quality in terms of DNA length. Additionally, short-read sequencing was performed on matched samples and quality control metrics were compared between the two sample types. Matched human tumors (n = 50) were processed either by formalin fixation and paraffin embedding or placed in cryovials containing cryopreservation medium. These cryovials were cooled slowly to -80°C and stored in liquid nitrogen. DNA was extracted using the same protocol for both tissue types with the exception that tissues embedded in paraffin were first dewaxed followed by a multistep rehydration protocol. Samples were weighed and calibrated for mass. After the column purification and elution, sample concentration and purity were measured. DNA fragment length was measured on a fragment microelectrophoresis analyzer. Graded amounts of tumor tissue were used to determine the lowest starting material needed to extract 200 ng of DNA. The average of all the samples reached the minimal threshold of 200 ng. However,74% of FFPE specimens failed to meet the minimum 40 ng/mg, whereas only 21% were below the threshold in the cryopreserved samples. In the cryopreserved group, the average DNA yield was 222.1 ng/mg, whereas 52.8 ng/mg was obtained from FFPE tissue. For DNA purity in cryopreserved tissues, the 260/280 ratio was 1.76, and in FFPE tissues the mean was 1.78. The DNA Quality Number (DQN) is a measurement of DNA fragment length and the percentage that exceeds the threshold of 300 bp. For FFPE, the DQN was 4.4 compared to a DQN of 9.8 for the cryopreserved samples. Setting a higher threshold of DNA length to 40,000 bp and measuring the area under the curve (AUC), it was observed that cryopreserved samples were 9-fold higher in fragments greater than 40,000 bp. Cryopreserved cancer tissue provides superior quality assurance measurements of DNA over FFFPE. Treatment decisions based on molecular results demand accuracy and validity. The pathology community should support efforts to cryopreserve cancer biospecimens in the clinical setting to provide valid molecular testing results. The automatic pickling of tumor specimens in formalin is no longer an acceptable default. Citation Format: Jared James Barrott, Nikole O'Neal, Jeffrey Okojie, David Booker, Ken Dixon. DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4957.

Abstract 2072: Human liver patient derived organoids: Development and characterization of a new tissue mimetic model

Abstract Liver is the largest solid organ and performs hundreds of vital functions including drug metabolism. Liver diseases, including viral infections, cancer and metabolic disorders account for about 2 million deaths annually, worldwide (1). Current models for studying liver biology and drug metabolism include 2D cultured primary cells/cell lines, which do not represent the metabolic and structural complexity of human tissue. In vivo models for liver diseases are time consuming, cost prohibitive and often ineffective. Therefore, better tissue mimetic liver models are needed. Organoids are self-assembled 3D cultured cellular units that may mimic the corresponding human tissue structurally and functionally (2). Patient derived organoids (PDO) are developed from tissue of specific patients and may resemble the source tissue in molecular features, including recapitulation of patient specific responses to therapies. Here, we report development and characterization of novel liver PDO lines from vendor-sourced cryopreserved tissue. We have developed biobanks of undifferentiated as well as differentiated liver PDO models.Human cadaver-sourced liver tissue were obtained with explicit donor consent. Liver PDO were generated following modification of a previously established protocol (3). Briefly, samples were minced, digested to single cells, and filtered. Specific number of cells were mixed with diluted Matrigel (Corning) and added as small drops on cell culture plates and grown in liver PDO specific growth media. Whenever appropriate, PDO were differentiated by culturing in differentiation-specific media in multi-well plates. In all cases, PDO were monitored using brightfield microscopy (Olympus CK40) and live cell imaging (Muvicyte, Perkin Elmer); and passaged every 6-8 days. Undifferentiated PDO were cryopreserved/thawed following in-house SOP to confirm viability. Liver-specific biomarker expression in the PDO lines were confirmed by confocal microscopy (ImageExpress, Molecular Devices). PDO lines were also assayed for ALT activity, albumin production and urea. PDO were cleared for microbial/viral contamination and confirmed as unique lines using single tandem repeat analyses. Transcriptome profiles were analyzed using vendor services. So far, we have successfully generated 3 liver PDO lines, both undifferentiated as well as differentiated. Our liver PDO lines would be highly useful in understanding liver biology and disease models; and will have significant usage in drug testing, including DMPK and ADME/Tox studies. Future efforts would include scaling up, development of liver specific assays and developing PDO from specific liver diseases, including hepatocellular carcinoma, MASLD and NASH.

Effect of cryopreservation on CD4+ T cell subsets in foreskin tissue.

Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations.

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue

PurposeSingle-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. So far, single-cell studies have been constrained by the requirement for prospectively collected fresh or cryopreserved tissue. This limitation might be overcome by recent technical developments enabling single-cell analysis of FFPE tissue.MethodsWe benchmark single-cell profiles from patient-matched fresh, cryopreserved and archival FFPE cancer tissue.ResultsWe find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathway, hallmark, and clinically useful signatures, although expression of single genes varied due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting.ConclusionOur analysis highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and increases the applicability in translational research.

A Low-Cost, Tabletop LOD-EPR System for Nondestructive Quantification of Iron Oxide Nanoparticles in Tissues.

Iron oxide nanoparticles (IONPs) have wide utility in applications from drug delivery to the rewarming of cryopreserved tissues. Due to the complex behavior of IONPs (e.g., uneven particle distribution and aggregation), further developments and clinical translation can be accelerated by having access to a noninvasive method for tissue IONP quantification. Currently, there is no low-cost method to nondestructively track IONPs in tissues across a wide range of concentrations. This work describes the performance of a low-cost, tabletop, longitudinally detected electron paramagnetic resonance (LOD-EPR) system to address this issue in the field of cryopreservation, which utilizes IONPs for rewarming of rat kidneys. A low-cost LOD-EPR system is realized via simultaneous transmit and receive using MHz continuous-wave transverse excitation with kHz modulation, which is longitudinally detected at the modulation frequency to provide both geometric and frequency isolation. The accuracy of LOD-EPR for IONP quantification is compared with NMR relaxometry. Solution measurements show excellent linearity (R2 > 0.99) versus Fe concentration for both measurements on EMG308 (a commercial nanoparticle), silica-coated EMG308, and PEG-coated EMG308 in water. The LOD-EPR signal intensity and NMR longitudinal relaxation rate constant (R1) of water are affected by particle coating, solution viscosity, and particle aggregation. R1 remains linear but with a reduced slope when in cryoprotective agent (CPA) solution, whereas the LOD-EPR signal is relatively insensitive to this. R1 does not correlate well with Fe concentration in rat kidney sections (R2 = 0.3487), while LOD-EPR does (R2 = 0.8276), with a linear regression closely matching that observed in solution and CPA.